1. The Field of the Invention
The present invention is directed generally to methods and compositions for active specific immunotherapy of tumors. More specifically the present invention is related to methods and compositions for treating tumors with vaccines and with preparations for intratumoral injections and to methods for preparing tumor vaccines and preparations for intratumoral injections that are capable of stimulating immune responses to specific tumor antigens.
2. The Relevant Technology
Basic terminology and general principles in immunology. The foundation of immunology theory rests on the basic idea of self/non-self discrimination, a process that is accomplished by means of recognition mechanisms. Because these recognition mechanisms are used for defeating undesirable microorganisms and for eliminating potentially harmful substances, they are in fact a preeminent part of an individual""s defense mechanisms.
Some of these defense mechanisms do not rely on the triggering effect of prior exposure for effecting their protective activity. These are part of the innate or natural immune mechanisms that include physical barriers like the skin, and certain substances, cells and enzymes that are active before exposure to foreign agents. In contrast, the defense mechanisms collectively known as acquired or adaptive immunity can recognize foreign structures and subsequent exposure to such foreign structures leads to a more efficient and effective immune response. See, for example, Donald M. Weir, and John Stewart, Immunology, chapter 1, 8th ed., Churchill Livingstone, New York, 1997. (This book will hereinafter be referred to as xe2x80x9cImmunologyxe2x80x9d).
A molecule that elicits a specific immune response when introduced into the host tissues is an antigen. Note that the definition of an antigen is arbitrary because specific responsiveness is a property of the host tissues, not of the injected substance. A response to stimulation by antigen is the production of an antibody, a protein that is capable of specific combination with antigen. See, for example, Immunology, chapter 3; The Dictionary of Immunology, edited by W. John Herbert, Peter C. Wilkinson, and David I. Scott, page 10, Academic Press, London, 1995. (This book will hereinafter be referred to as xe2x80x9cDictionary of Immunologyxe2x80x9d). The response of the host to an antigen on the first encounter is termed the primary immune response. Dictionary of Immunology, at p. 132.
The self/non-self discrimination idea in immunology should not be confused with the external/internal dichotomy. It is worth emphasizing in this respect that the physiological reaction that develops through an antibody response is triggered by a foreign agent, whether this agent is harmful or not. In addition, the foreign agent does not necessarily have to be external, for immune responses can also be generated against internal antigens. Sources of such internal antigens include antigens released from disintegrating tissues and antigens that are produced during replicative cycles. Immunity is achieved when the necessary antigen is present and induced antibodies can neutralize the foreign agent. See, for example, Immunology at pp. 86-87. Humoral immunity depends on the appearance in the blood of proteins such as those known as antibodies.
For self-non-self discrimination, especially in the context of tumor immunology, cellular immunity is much more important than antibodies. Cellular immunity, also known as cellular cytotoxic immunity or cell-mediated immunity, was originally used to describe localized reactions to organisms mediated by a type of lymphocytes, the T-lymphocytes, and by phagocytes rather than by antibody. It is currently used to describe any response in which antibody plays a subordinate role. It is acknowledged that cell-mediated immunity depends mainly on the development of T-cells that are specifically responsive to the inducing agent, and furthermore cell-mediated immunity is generally active against intracellular organisms. See, Immunology, pp. 86-87.
Adjuvants. The natural ability of an antigen to induce an immune response can be modified, and in particular enhanced, by altering or by mixing it with another substance. The procedure or the substance used to enhance immune responses is called an adjuvant. At least three classes of adjuvants have been used for a long time; these are mineral oil emulsions, aluminum compounds, and surface active materials such as saponin, lysolecithin, retinal, Quil A(copyright), some liposomes, and pluronic polymer formulations. See, for example, Fundamental Immunology, edited by William E. Paul, at p. 1008, Raven Press, New York (this book will hereinafter be referred to as xe2x80x9cFundamental Immunologyxe2x80x9d). Aluminum adjuvants used alone or in combination include aluminum hydroxide gel, aluminum phosphate, aluminum sulphate, and alums comprising ammonium alum (such as (NH4)2SO4.Al2(SO4)3) and potassium alum. Aluminum hydroxide (hereinafter xe2x80x9cALxe2x80x9d) is one of the older adjuvants and it is considered so safe that it has been applied in bacterial and viral vaccines administered to billions of people around the world. Calcium phosphate gel (hereinafter xe2x80x9cCPxe2x80x9d) has similar properties and is also used in vaccines. Both substances are available in pharmaceutical qualities in most countries worldwide. Techniques for preparing adjuvant-antigen preparations for injection are well known in the art. See, for example, Terry M. Phillips, Analytical Techniques in Immunochemistry, pp. 307-10, Marcel Dekker, New York, 1992.
Other adjuvants include complete Freund""s adjuvant (a water-in-oil emulsion in which killed, dried, mycobacteriaxe2x80x94usually M tuberculosisxe2x80x94are suspended in the oil phase); incomplete Freund""s adjuvant (analogous to the complete Freund""s adjuvant with no mycobacteria); ISCOM (or immune stimulating complex, comprising lipophilic particles formed by the spontaneous association of cholesterol, phospholipid and the saponin Quil A(copyright)); lipopolysaccharide (complex molecules consisting of a lipid corexe2x80x94lipid Axe2x80x94with a polysaccharide side chain that are components of certain bacilli, Lipid A is incorporated into the outer membrane of the bacterium and the polysaccharide projects extracellularly. Their adjuvant potency is associated with lipid A; they are also mitogenic for murine B lymphocytes); and mycobacterial adjuvants (whole, heat killed, dried, mycobacteriaxe2x80x94such as M. tuberculosis, M. avium, M. phlei, and M. smegmatis) that, when suspended in mineral oil and emulsifier, have adjuvant activity with respect to any antigen given with them. Extracts of some mycobacteria, e.g., mycobacterial peptidoglycolipids have similar adjuvant activities. See, for example, Dictionary of Immunology at pp. 3, 7, 46, 94, 97, 105, and 116; R. B. Luftig, Microbiology and Immunology, pp. 228-29, Lippincott-Raven Publishers, Philadelphia 1998. Microbial adjuvants include Corynebacterium parvum and Bordetella pertussis. See, for example, Handbook of Immunology at 115-16. Use of controlled-release preparations and materials with adjuvant activity and possible sites of action have been described in Fundamental Immunology at pp. 1007-09.
Mineral carriers such as aluminum hydroxide, potassium ammonium sulphate, and potassium aluminum sulphate adsorb the antigen on their surface. These common adjuvants have been used at a 0.1% concentration with up to 1 mg protein antigen in 1 ml administered to animals at doses of 0.2-0.5 ml/(kg body weight). See Miroslav Ferencik, Handbook of Immunochemistry, p. 115, Chapman and Hall 1993 (this book will hereinafter be referred to as xe2x80x9cHandbook of Immunochemistryxe2x80x9d). Although Freund""s adjuvant is toxic and not used for immunization of human beings, mineral adjuvants such as aluminum hydroxide are common in human medicine. Id. at 116. In addition to alum, other adjuvants in the group of inert carriers include bentonite, latex, and acrylic particles. See Fundamental Immunology at 1008.
Combinations of adjuvants can also have adjuvant properties. For example, it has been shown that the combination of saponin and muramyl dipeptide in a squalene in water emulsion is superior to alum as an adjuvant for inducing certain responses in mice. R. Bomford, M. Stapleton, S. Wilson, A. McKnight, and T. Andronova, The control of the antibody isotype responses to recombinant human immunodeficiency virus gp120 antigen by adjuvants, AIDS Res. Hum. Retroviruses Vol. 8(1992) pp. 1765 et seq. These adjuvants are complemented by new adjuvants that have been developed during the last fifteen years. See, for example, Anthony C. Allison, The Role of cytokines in the Action of Immunological Adjuvants, in Vaccine Design. The Role of cytokine Networks, edited by Gregory Gregoriadis and Brenda McCormack, NATO ASI Series A: Life Sciences Vol 293, pp. 1-9, Plenum Press, New York 1997 (this book will hereinafter be referred to as xe2x80x9cVaccine Designxe2x80x9d); Immunology at p. 116; H. Snippe, I. M. Fernandez and C. A. Kraaijeveld, Adjuvant Directed Immune Specificity at the Epitope Level. Implicationsfor Vaccine Development. A Model Study Using Semliki Forest Virus Infection of Mice, in Vaccine Design at pp. 155-73.
An adjuvant can be administered prior to, simultaneously with, or following the administration of the antigen. Antibody production enhancement caused by adjuvants is not fully understood. However, adjuvant properties that may exist either alone or in various combinations and which permit a substance or formulation to be described as adjuvant active have been defined. See, for example, J. C. Cox and A. R. Coulter, Adjuvantsxe2x80x94A classification and review of their modes of action, Vaccine Vol. 15(1981) pp. 248 et seq.; John Cox, Alan Coulter, Rod Macfarlan, Lorraine Beezum, John Bates, Tuen-Yee Wong and Debbie Drane, Development of an Influenza-ISCOM(trademark) Vaccine, in Vaccine Design at pp. 33-49. One of these properties is depot generation, whereby the vaccine is retained near the dose site to give short term trickle release or a longer term pulsed release. Id. at p. 34. The term xe2x80x9cdepotxe2x80x9d will hereinafter be used to refer to an adjuvant or to the combination of an adjuvant and at least one immunostimulating substance that is administered with antigenic material for enhancing the immune response.
Lymphocytes and cytokines. The immune system in the higher animals comprises a collection of organs and cell types, and all these cells develop from stem cells in tissues such as the bone marrow. One of these types of cells in particular, the white blood cells or leucocytes, are produced through two main pathways of differentiation. More specifically, the lymphoid lineage is the differentiation pathway that leads to the production of T-lymphocytes (also known as T cells) and B-lymphocytes, collectively lymphocytes. These are cells that are found, inter alia, at sites where immune responses are taking place. More specifically, a lymphocyte is an antigen-receptor carrying cell that recognizes the antigen, effectively embodying a mediator cell of specific immunity. See, for example, Immunology at pp. 8-9, Dictionary of Immunology at pp. 107, 147.
In addition to cells, many secreted molecules, known as cytokines, play a role in the different phases and aspects of immune responses. Cytokines are usually glycoproteins made and secreted by cells and they act as cellular mediators with effects on the same or other cells"" characteristics such as growth, differentiation, and activation. More generally, cytokines are defined as regulatory peptides that can be produced by virtually every nucleated cell type in the body. See, for example, Joost J. Oppenheim, Foreword, in The Cytokine Handbook, edited by Angus Thomson, pp. xviii-xxii, Academic Press, Norfolk, UK 1998 (this book will hereinafter be referred to as xe2x80x9cCytokine Handbookxe2x80x9d); Barbara Detrick and John J. Hooks, Cytokines in Human Immunology, in Handbook of Human Immunology, edited by Mary S. Leffell, Albert D. Donnenberg, and Noel R. Rose, pp. 233-66, CRC Press, Boca Raton, Florida 1997 (this book will hereinafter be referred to as xe2x80x9cHuman Immunology Handbookxe2x80x9d). The term interleukin (hereinafter abbreviated as xe2x80x9cILxe2x80x9d) is part of the designations of a number of cytokines. See, for example, Jan Vilcek, The Cytokines: An Overview, in Cytokine Handbook, pp. 1-33.
It is currently suspected that some cytokines such as the Colony Stimulating Factors (xe2x80x9cCSFxe2x80x9d), might have some properties that relate to long-range effects in addition to the shortrange effects that are acknowledged as more typical of cytokines. Nevertheless, the main function of most cytokines appears to be paracrine, this term referring to the signaling to or attracting of other lymphocytes in proximity. Some cytokines, and in particular IL-2, also have an autocrine function, this term referring to self stimulation that is derived from the cytokine binding to receptors on the same cell that previously secreted the cytokine.
The name interleukin is given to certain cytokines that act as intercellular signals. Nevertheless, it is accepted that there is no logic to the interleukin designation and there is no logic to the order in which the interleukins are numbered either. For example, cytokines such as certain interferon or tumor necrosis factor could also be designated with the term interleukin. Dictionary of Immunology, at p. 96. As of 1996, the interleukin series had reached 18. Cytokine Handbook, at p. 3.
The cytokines that are produced by lymphocytes are termed lymphokines and their release is often stimulated following contact with antigens. Cytokines such as IL-2 and IL-4 are lymphokines. IL-2""s major function is in the regulation of the immune response. See, for example, Immunology, at pp. 86-146. Background material on IL-2 can be found in, for example, Kendall A. Smith, Interleukin-2: Inception, Impact, and Implications, Science Vol. 240 (1988), pp. 1169-76; Sarah L. Gaffen, Mark A. Goldsmith, and Warner C. Greene, Interleukin-2 and the Interleukin-2 Receptor, in Cytokine Handbook at pp. 73-103; Christopher J. Secombes, The Phylogeny of Cytokines, in Cytokine Handbook at pp. 965-71; Interleukin-2, edited by Kendall A. Smith, Academic Press, San Diego, Calif. 1988 (this book will hereinafter be referred to as xe2x80x9cInterleukin-2xe2x80x9d); Robin Thorpe, Interlekin-2 in Cytokines, edited by Anthony Mire-Sluis and Robin Thorpe, pp. 19-33, 526-27, Academic Press, San Diego, Calif. 1998 (this book will hereinafter be referred to as xe2x80x9cCytokinesxe2x80x9d). Background material on other interleukins can be found in, for example, Cytokine Handbook at pp. 35-72, 105-499, and background material on other cytokines can be found in, for example, Cytokine Handbook at pp. 491-823, 885-993, and in Cytokines at pp. 1-18, 35-546. It is acknowledged that no short definition can encompass all the essential properties of cytokines, which are better defined by a set of characteristic features. See, for example, Cytokine Handbook at p. 4. Despite the existence of these characteristic features, individual cytokines, and in particular individual interleukins, fall into different families when they are classified according to features such as structure, receptors, and stimulatory and inhibitory actions. Furthermore, a plurality of synergistic and antagonistic interactions among cytokines have been reported. See, for example, Cytokine Handbook at pp. 6-14.
Methods for the measurement and detection of cytokines are described in, for example, Meenu Wadha and Robin Thorpe, Assays for Cytokines, in Cytokine Handbook at pp. 855-84. IL-2, formerly known as T cell growth factor, is an immunostimulant. More precisely, by the application of IL-2 the immune system can be stimulated to become active against tumor cells. Production and characteristics of natural and recombinant human IL-2 have been described by K. Kato, Characteristics of Natural and Recombinant Human Interleukin 2, in Interleukin-2 at pp. 37-66 and references therein. It is known that recombinant human IL-2 reacts in the same manner in human beings as it does in mice.
Immunomodulators, of ten contained in adjuvants, induce the production of cytokines, thus enhancing immune responses. Examples are muramyl peptides, lipopolysaccharides and derivatives, and certain cationic detergents. See, for example, Anthony C. Allison, The Role of Cytokines in the Action of Immunological Adjuvants, in Vaccine Design at pp. 1-9. Interleukin active domains or the corresponding synthetic peptides could in fact be potent adjuvants, as shown for a region of an IL-1. See, for example, Aldo Tagliabue and Diana Boraschi, Interleukin 1 and Its Synthetic Peptide 163-171 as Vaccine Adjuvants, in Vaccine Design at pp. 167-73.
Immunization and Immunology. Specific immunity is generally understood as a developed non-susceptibility to re-infection by a pathogen, thus implying survival after prior exposure to the same pathogen. In other words, specific immunity results from the recognition of antigen/cell that leads to the production of specific antibody and/or the stimulation T-lymphocytes that subsequently specifically react with the recognized antigen/cell. See, for example, Dictionary of Immunology at 147.
It follows from the foregoing Concise overview of basic terminology and concepts in immunology that acquiring immunity to threatening agents is a desirable goal in an organism""s overall defense strategy. Immunization provides the line of defense that relies on acquired immunity. The mechanism of this form of immunity involves the contact between the antigens of the invading agent and the immune system cells, including lymphocytes, followed by an immune response that is specific to the foreign agent.
Immunization is the process by which specific immunity is induced as a preventive measure in the fight against many diseases. Immunization is a general term, and the term vaccination is used when patients are immunized. In general, immunization can be used as a preventive or as a therapeutic treatment. The preventive use of immunization is a prophylactic treatment, whereas the use of immunization while the disease is in progress is immunotherapy. Immunization provides two types of acquired immunity, active and passive. Immunotherapy is the treatment of a disease by immunization, active or passive, or by the use of agents that modify the actions of lymphocytes. In particular, immunotherapy refers to the stimulation of the immune system and conventionally uses a form of immunostimulant, a substance that causes a general, non-specific, stimulation of the immune system. The American Medical Association Encyclopedia of Medicine, p. 576 (this encyclopedia will hereinafter be referred to as xe2x80x9cAMA Encyclopedia of Medicinexe2x80x9d).
Passive immunization involves the transfer of pre-formed antibodies that provide immediate, short lived, protection against specific disease-causing cells. Passive vaccination typically involves the administration of either serum of an immune individual into another individual that might be infected, or antibodies that are purified from such toxin-immune sera. Active immunization primes the body to make its own antibodies against disease causing agents/cells and confers longer lasting immunity. This priming can be accomplished by overt clinical infection, inapparent infection or deliberate artificial immunization. Whereas low levels of antibody are characteristically produced slowly during the primary immune response, priming of the tissues in which the predominant cells are lymphocytes or lymphoid tissues allows a secondary immune response to be evoked on or subsequent challenge. When the secondary immune response takes place, there is a very rapid production of large amounts of antibody over a few days followed by a slow exponential fall. This pattern is common to a plurality of immune response mechanisms. See, for example, Dictionary of Immunology, at pp. 142.
Vaccination is a form of deliberate artificial immunization whereby antigenic material, or vaccine, is administered. The administered antigens can be in the form of killed or weakened cells that sensitize the immune system such that if disease causing cells with the same antigen later enter the body, they are quickly destroyed. See, for example, Immunology, at pp. 87-88; AMA Encyclopedia of Medicine at 573-574 and 1034; S. J. Cryz, Jr., in Immunotherapy and Vaccines, edited by Stanley J. Cryz, pp. 3-11, VCH, Weinheim, Germany 1991. For an overview of the immune system from a molecular perspective, see, for example, Mary S. Leffell, An Overview of the Immune System: The Molecular Basis for Immune Responses, in Human Immunology Handbook pp. 1-45.
In general, the following materials are used for vaccination: live bacteria or viruses (dangerous, e.g., variola vacciniae against smallpox); killed bacteria or viruses (sometimes with dangerous side effects); weakened viruses (e.g., for vaccination against poliomyelitis); attenuated bacteria or viruses (similar to weakened viruses); viruses that affect animals and make them sick or kill them but that are harmless to humans or just make them slightly sick, such as the vaccinia cowpox virus; parts of bacteria such as antigens and membrane fractions, also known as cellular vaccines, which are much safer but sometimes not as effective (e.g., Bordetella pertussis), and if bacteria are pathogenic due to the toxins that they release, then toxoids or inactivated toxins can be used in vaccination (e.g., Diphtheria toxin and tetanus toxin (from clostridium tetani)).
Sometimes the antibodies and cells that are induced against an animal virus also react with a similar human virus. The immune response is then termed cross-reactive. For example, the immune response induced against cowpox virus, which is genetically similar to the human pathogenic smallpox virus, is cross-reactive.
When any biological activity of the antigenic material to be administered is destroyed prior to its administration, the antigenic material is incapable of replication, the vaccine that contains such material is called an inactivated vaccine, and the destructive process is called inactivation. For example, irradiation of cells with an appropriate dose of X-rays of fers one way of inactivating the disease-causing aspects of the irradiated cells while they retain their antigenicity and morphology. The cells so irradiated are thus capable of promoting an immune response, but they are not capable of causing disease. In contrast, a live vaccine that contains organisms or viruses that have been cultured or otherwise treated under conditions in which they lose virulence but retain the capacity to stimulate an immune response is called an attenuated vaccine and the process that renders the antigenic material under such conditions is called attenuation. Attenuation reduces the ability to cause disease, but it does not significantly alter antigenicity. See, for example, Dictionary of Immunology, at pp. 17, 93.
Immunology and cancer prevention and therapy. Historical records reveal that strategies for acquiring immunity against threatening agents have been pursued for a long time, although with varying degrees of understanding of the mechanisms involved. In particular, records of certain forms of immunization have been traced back to the sixth century in China, where at least immunization against smallpox was practiced at about AD 590 and it has been reported that this form of immunization was also practiced in India in ancient times. See, for example, Immunology at 4-5. With an ever increasing understanding derived from the intense use of modern clinical and biochemical research tools, immunization is actively pursued; acquiring immunity against certain threatening agents is viewed nowadays as one of the potentially more successful means for defeating such agents. See, for example, Philip Livingston, Conference Overview, in Specific Immunotherapy of Cancer With Vaccines, edited by Jean-Claude Bystryn, Soldano Ferrone, and Philip Livingston, Annals of the New York Academy of Sciences Vol. 690, pp. 1-5, New York 1993 (this publication will hereinafter be referred to as xe2x80x9cImmunotherapy of Cancer With Vaccinesxe2x80x9d). One of the agents against which the effect of immunological preventive and therapeutic techniques have been incessantly studied is cancer. See, for example, Ingegerd Hellstrom and Karl Erik Hellstrxc3x6m, Tumor Immunology: An Overview, in Immunotherapy of Cancer With Vaccines, at pp. 24-33.
A lesson learned from the use of vaccines against infectious diseases is that vaccines do not prevent infection; instead, they limit it. Once the vaccine has primed the immune system, natural systems and local immunity cure infection at the site of entry. If circulating tumor cells can be eliminated by the appropriate antibodies or by immune system cells, local treatment may cure cancer much like vaccination leads to the defeat of infectious diseases.
One of the problems presented by the treatment of human cancers with vaccines, however, is that the immunogenicity of tumor antigens is relatively low. Immunogenicity is the potential of an antigen to stimulate an immune response. In general, tumor antigens are not sufficiently immunogenic to induce more than occasional immune responses. Immunotherapy of Cancer With Vaccines, at p. 4. This fact notwithstanding, a tumor""s low immunogenicity in standard immunization experiments does not necessarily mean that it lacks molecules that can, under appropriate conditions, be recognized as antigenically foreign. Concisely put, antigenicity is the capacity of a substance to act as an antigen and consequently, immunogenicity and antigenicity are separate characteristics. This difference is illustrated by the fact that antigenic tumors of ten escape immunologic control. Id. at pp. 26-27. In the context of this specification, xe2x80x9cimmunogenicityxe2x80x9d is used to characterize tumor cells or proteins that are used for inducing immune responses and xe2x80x9cantigensxe2x80x9d describes materials used for testing, demonstrating or measuring immune responses. For an overview on immunity to tumors and tumor antigens see, for example, Immunology at 258-66.
The low immunogenicity of tumor antigens can be concisely and generally explained as follows. In contrast to bacteria or viruses, which invade the organism from the outside world, tumors are derived from the organism""s own cells by mechanisms only partially understood today. Consequently, they bear surface structure, which is derived from the organism""s repertoire of xe2x80x9cself antigensxe2x80x9d and they are tolerated by the host""s defense systems. When such tumors have been growing in an organism for a long time (perhaps years or even decades) they develop an increasing number of foreign characteristics. This is due to the many cellular divisions that they undergo which in turn leads to a rather chaotic genetic organization. Tumors might therefore be eventually very different from the host""s cells. However, due to their slow growth and other unknown factors, the host""s immune system gets accustomed to the more and more foreign-looking tumor cells and it does not react to them. xe2x80x9cTumor-specific antigensxe2x80x9d are a rarity. Only a few such antigens are known, among them the idiotypes of antibodies in the membranes of B lymphoma cells. Most of the tumor antigens are self-antigens or altered self-antigens, or antigens that belong to the repertoire of self-structures. In addition, tumors might expose antigens that have been expressed only during embryonal or fetal development and that might, therefore, be xe2x80x9cunknownxe2x80x9d to the immune system. These types of antigens include onco-fetal antigens and carcino-embryonal antigens, such as alpha fetoprotein (AFP) or carcino embryonic antigen (CEA). Tumor cells might also be weak antigens because they miss some of the normal antigenic structures that are needed to recognize a cell as foreign, such as the MHC antigens.
Active specific immunotherapy approaches to the treatment of tumors have been widely investigated during recent years. Numerous studies involving the vaccination of patients with their own inactivated tumor cells have been reported. These studies have demonstrated that inclusion of an adjuvant is necessary to stimulate the patient""s immune system against the autologous, or derived from self, tumor cells. For example, methods utilizing the particulate adjuvant, Bacillus Calmette-Guerin (BCG) cells, administered systemically or mixed with the patient""s own tumor cells have been shown to induce tumor-specific immunity in laboratory animals. Peters, L. C., Brandhorst, J. S., Hanna Jr., M. G., Preparation of Immuno-Therapeutic Autologous Tumor Cell Vaccines from Solid Tumors; Cancer Res. 39: 1353-1360 (1979).
This approach has been investigated with different tumor types. The administration of inactivated tumor cells in a mixture including a bacterial adjuvant resulted in significantly improved survival rates in patients with metastasized renal cell carcinoma. Tallberg, T., Tykkxc3xa4, H., Specific Active Immunotherapy in Advanced Renal Cell Carcinoma: A Clinical Long-Term Follow-Up Study; World J Urology 3: 234-44 (1986). A statistically significant increase in patient survival rates was also achieved with the use of Newcastle Disease Virus (NDV) as an adjuvant in clinical trials for the treatment of malignant melanoma. Cassel, W. A., Murray, D. R., Phillip, H. S., A Phase II Study on The Post Surgical Management of Stage II Malignant Melanoma With a Newcastle Disease Virus Oncolysate, Cancer 52: 856-60 (1983). A similar protocol employed in animal tumor models demonstrated that administration of a vaccine prepared by irradiating ESb tumor cells infected with the apathogenic NDV induced permanent T-cell immunity towards antigens of the introduced tumor type. Schirrrnacher, V., Ahlert, T., Heicappel, R., Appelhans, B., Von Hoegen, P., Successful Application of Non-Oncogenic Viruses for Antimetastatic Cancer Immunotherapy; Cancer Reviews 5: 19-49 (1986); Schirrmacher, V., Immunity and Metastasis: In Situ Activation of Protective T Cells by Virus-Modified Cancer Vaccines; Cancer Survey 13: 139-154 (1992). Moreover, the immunity was transferable to other animals by adoptive transfer of lymphocytes. Schirrmacher, V. Von Hoegen, P. Griesbach, A., Zangemeister-Wittke, U., Specific Eradication of Micrometastasis by Transfer of Tumor-Immune T-Cells From MHC-Congenic Mice; Cancer Immunol. and Immunother. 32: 373-81 (1991).
NDV, however, is not a conventional adjuvant. During incubation with tumor cells the fowl-pathogenic and human apathogenic virus binds to the tumor cells and the virus membrane gets integrated into the tumor cells membrane which by this gets a certain degree of xenogenization. In addition, the virus membrane contains components (hemagglutinins) to which human (and animal) cells (lymphocytes) can bind. It is anticipated that the immune system is stimulated by the xenogenization of the tumor cell membranes altered by the interaction of the virus membrane. In addition, lymphocytes can bind to the hemagglutinin moiety the tumor cells have acquired from the virus and become stimulated upon binding. This might also render the otherwise non-immunogenic parts of the tumor cells more immunogenic.
Regarding the use of adjuvants in cancer treatment, one may think of using a substance with well-known adjuvant properties such as aluminum hydroxide. This substance is used in bacterial and viral vaccines for inducing humoral immunity, which is antibody-based immunity. However, it is acknowledged that the induction of humoral immunity responses is or can be counterproductivexe2x80x94depending on the type of tumorxe2x80x94in tumor therapy. It has been shown that antibodies to tumor antigens might mask the tumor antigens and thus protect the tumor from the desired aggression by T-lymphocytes and other immune system cells. Due to this known property of inducing immune responses that might even protect the tumor from the attack of the cellular immune system, most researchers have ignored aluminum hydroxide and have not investigated its properties as an adjuvant for tumor vaccines. Those who have investigated these properties in animal experiments have clearly shown that aluminum hydroxide in fact does not have adjuvant activity at all. See, B. E. Souberbielle, B. C. Knight, W. J. Morrow, D. Darling, M. Fraziano, J. B. Marriott, S. Cookson, F. Farzaneh, and A. G. Dalgleish, Comparison of IL-2 and IL-4-transfected B16-F10 cells with a novel oil-microemulsion adjuvant for B16-F10 whole cell tumor vaccine, Gene Therapy Vol. 3 (1996) 853-858. Consistently with this knowledge, some researchers have ceased using aluminum hydroxide in experimental tumor vaccination. In addition, it has been shown that aluminum hydroxide does not act as an adjuvant when injected together with tumor cells or when injected alone. This knowledge is supplemented by the already reported property of aluminum hydroxide as an adjuvant only for soluble protein or for carbohydrate antigens, not for cells.
Cytokines and cancer immunotherapy. Clinical trials with IL-2 have shown on the one hand that cytokines can stimulate immunity and lead to complete tumor regression in some patients. On the other hand, it has also been shown that systemic therapy with cytokines can be extremely toxic, thus limiting its effectiveness. See, for example, John A. Sogn, John F. Finerty, Anne K. Heath, Grace L. C. Shen, and Faye C. Austin, Cancer Vaccines: The Perspective of the Cancer Immunology Branch, NCI, in Immunotherapy of Cancer With Vaccines at p. 326. Furthermore, it is known that xe2x80x9cdetermining the best cytokine or cytokines to use is difficult because so many cytokines have the potential to augment immunity and because virtually all of the cytokines tested in mice have shown some potential usefulness.xe2x80x9d Id. at p. 327.
It is known that administration of IL-2 to patients is of ten associated with adverse effects, sometimes so severe that the therapy must be halted. The complications include the development of severe vascular permeability which leads to interstitial pulmonary edema and eventual multiorgan failure if the therapeutic administration of IL-2 is not reduced or discontinued. A mouse study has characterized this condition and the IL-2-induced formation of certain products is believed to be involved in the process. Although less frequently, other adverse effects observed during or following the administration of IL-2 include cardiomyopathy, scleroderma, myelodysplasia, hypothyroidism, diabetes, renal disease, colonic ischemia, inflammatory arthritis, hypoprothrombinemia, fever, diarrhea, and asthemia. In addition, patients may develop antibodies against IL-2 that could compromise therapy. Given this sequel of adverse effects, it is acknowledged that xe2x80x9c[t]herapeutic strategies, including dosing and route of administration, are largely influenced by attempts to limit adverse effects associated with IL-2 therapyxe2x80x9d. Robin Thorpe, Interleukin-2, in Cytokines at p. 27.
Cytokines have been reported as having generally potent effects on the development of the immune response to tumors and as eliciting a response capable of rejecting tumors. See, for example, A. McAdam, B. Pulanski, S. Harkins, E. Hutter, J. Frelinger, and E. Lord, Coexpression of IL-2 and xcex3-IFN Enhances Tumor Immunity, in Immunotherapy of Cancer With Vaccines at p. 349. Cytokines have also been used in vaccines for humoral immune responses. See, for example Dragana Jankovic, Patricia Caspar, Martin Zweig, Maria Garcia-Moll, Stephen D. Showalter, Frederick R. Vogel, and Alan Sher, Adsorption to Aluminum Hydroxide Promotes the Activity of Il-12 as an Adjuvant for Antibody as Well as Type 1 Cytokine Responses to HIV-1 gp 120, The Journal of Immunology Vol. 159 (1997) pp. 2409-17, at p. 2412. Although cytokines may play crucial roles in therapeutic vaccines for cancer treatment, these observations require a call for caution xe2x80x9cbecause cytokines have as much potential to stimulate tumor growth as to retard it, and many cytokines effectively suppress immune responses under some conditionsxe2x80x9d; it is further acknowledged that these xe2x80x9ccomplexities can only be unraveled by additional animal studies and direct testing in humans of promising candidate cytokines.xe2x80x9d John A. Sogn, John F. Finerty, Anne K. Heath, Grace L. C. Shen, and Faye C. Austin, Cancer Vaccines: The Perspective of the Cancer Immunology Branch, NCI, in Immunotherapy of Cancer With Vaccines, at p. 327.
In particular, numerous studies have utilized interleukin-2 (IL-2) in the form of recombinant IL-2 (hereinafter referred to as xe2x80x9crIL-2xe2x80x9d) which became available in therapeutically meaningful quantities as a result of genetic engineering techniques. Although systemic application of rIL-2 demonstrated promising therapeutic effects, severe and occasional lethal side effects were produced. Forni, G., Siovarelli, M., Santoni, A., Modesti, A., Forni, M., Interleukin-2 Activated Tumor Inhibition In Vivo Depends on the Systemic Involvement of Host Immunoreaetivity: J Immunol. 138: 4033-41 (1987); Waldmann, T. A., The IL-2/IL-2 Receptor System: A Target for Rational Immune Intervention; Immunol. Today 14: 264-70 (1993). In addition, the costs associated with this therapy can be very high.
The severe side effects may be due to the magnitude of the systemic doses of cytokines which must be administered to achieve the therapeutic benefits. Cytokines facilitate communication between cells. During such cell-to-cell communications, high concentrations are achieved at a localized cellular level but the concentration of circulating cytokines is typically very low. More specifically, if a lymphocyte meets a non-self antigen, for example, a tumor cell, it signals to other cells of the immune system about this encounter by secretion of cytokines. It was shown that the local concentration of IL-2 secreted by an activated T-lymphocyte is very high. The concentration of IL-2 in the secretory granules in which IL-2 is stored is in the range of 1-100 mM, which is a very high local concentration (equivalent to about 10-1000 g/l). See, D. R. Kaplan, Autocrine secretion and the physiological concentration of cytokines, Immunology Today Vol. 17 (1996) pp. 303-304. After release of IL-2 into the local environment, the local concentration remains very large, but this concentration is very small just a few cell diameters away from the release site. Consequently, a cytokine concentration gradient develops in the neighborhood of the cytokine release site, which is where the encounter with the antigen has taken place. Released cytokine molecules diffuse and finally reach the vascular system, where lymphocytes will detect the cytokine and migrate according to the cytokine concentration gradient towards regions with increasingly higher cytokine concentration, eventually reaching the site of the encounter with the antigen, invading it, secreting additional cytokines, and thus attracting greater numbers of lymphocytes to the site. This is in essence a chemotactic reaction of lymphocytes to cytokine concentration gradients.
Systemic delivery of a cytokine, however, results in xe2x80x9cfloodingxe2x80x9d of the entire organism with concentrations which would normally be present only at a localized cellular level. Consequently, immune system cells cannot locate the source of the cytokines that were released upon reaction with the antigenic material, which may further lead to the suppression of the beneficial effects of cytokines or even to the failure of the immune system. Local administration of cytokines has been investigated with variable results. Local administration in the form of an inhaled spray for the treatment of lung metastases of renal cell carcinomas appears to permit sufficient quantities of cytokines to be retained in proximity to the tumor lesions. Other attempts to localized administration, however, have been less successful. Because of their small size, the cytokine molecules tend to diffuse away from the application site and into surrounding tissue or be carried away and diluted in body fluids. In addition, the very short half-life of cytokines in blood results in a very short period of activity.
Concisely, the administration of cytokines may lead to complications and deleterious effects. It is therefore desirable to find the dosage that can be tolerated by living beings without causing undesirable side effects.
Cancer immunotherapy and localized delivery of cytokines. Numerous attempts have been made to direct the immunostimulating activity of cytokines to the tumor site. Some of these attempts rely on transfection techniques, transfection being the artificial transfer of foreign DNA into a eukaryotic cell. In animal models, this local cytokine immunostimulating activity has been achieved through the method of transfecting cytokine encoding genes into the tumor cells thereby causing the tumor cells to produce and secrete the cytokines. Cytokine gene-transfected cells contain the cytokine source and also the source of the immunogenic material. Karasuyama, H., Melchers, F., Establishment of Mouse Cell Lines Which Constitutively Secrete Large Quantities of interleukin 2, 3, 4, or 5, Using Modified cDNA Expression Vectors; Eur. J. Immunol. 18: 97-104 (1988).
Additional research into this promising method has been performed involving most of the known cytokine genes and various animal tumor models, especially mouse models. Because intact viable tumor cells, capable of multiplying, are necessary for the expression of the transfected cytokine gene, this method requires the use of viable tumor cells in the vaccine. Following injection into the experimental animal, the viable cytokine-gene-transfected tumor cells initially replicate to form a tumor. Over a period of time, typically 1-2 weeks, however, the tumor begins to diminish and eventually disappears entirely. Appearance of systemic cytokine activity follows the same pattern. Animals subjected to this treatment subsequently demonstrate immunity to xe2x80x9cre-infectionxe2x80x9d by tumor cells of the original genotype but not to other tumor types. See, e.g., Ley, V., Roth, C., Langlade-Demoyen, P., Larsson-Sciard, E. L., Kourilsky, P., A Novel Approach to the Induction of Specific Cytolytic T Cells In Vivo, Res. Immunol. 141: 855-63 (1990).
Further experiments have shown that immunity to non-transfected and, thus, non cytokine-producing, tumor cells can be induced by administering these tumor cells closely mixed with cytokine-producing tumor cells. Pardoll, D., New Strategies for Active Immunotherapy With Genetically Engineered Tumor Cells; Curr. Opin. Immunol. 4: 619-623 (1992). The induction of immunity appears to require that both the antigenic stimulus of the non-transfected tumor cells and the immunostimulus of the cytokine-producing cells originate from the same location within the experimental animal although not necessarily from the same cell. Further support for this theory arises from experiments in which immunity was induced by the administration of target tumor cells accompanied by nontransformed somatic cells, typically fibroblasts, transfected with a cytokine gene. See, e.g., Sobol, R. E., Fakhrai, H., Gjerset, R., Active Tumor Immunotherapy With Transduced Fibroblasts; Protocols Amer. Assn. Cancer Res. 33: 495-502 (1992).
Simultaneous exposure to multiple cytokines has been shown to be an especially effective method of inducing immunity with gene-transfected tumor cells. Vaccination of mice with a mixture of IL-2-gene-transfected and IL-4-gene-transfected tumor cells induced stronger immunity than vaccination with cells transfected with only one or the other of the cytokine genes. Ohe, Y. Podack, E. R., Olsen, K. J. et al., Combination Effect of Vaccination With IL-2 and IL-4 cDNA Transfected Cells on the Induction of a Therapeutic Immune Response Against Lewis Lung Carcinoma Cells; Intl. J. Cancer 52: 432-37 (1993). Tumor cells transfected with both IL-2 and Tumor Necrosis Factor (TNF) cytokines were more effective at inducing immunity than cells infected with only one of the two genes. Ohira, R., Ohe, Y., Heike, Y., Podack, E. R., Olsen, K. J., Nishio, K., Nishio, M., Miyahara, Y., Funayama, Y., Ogasawara, H., Arioka, H., Kato, H., Saijo, N., Gene Therapyfor Lewis Lung Carcinoma With Tumor Necrosis Factor and Interleukin-2 cDNAs Co-Transfected Subline; Gene Therapy 1: 269-275 (1994).
It is also known that lymphocytes obtained from cancer patients that were activated with IL-12 and IL-2 revealed greatly augmented cytotoxicity against autologous tumor cells compared with that induced by IL-2 alone. See, for example, Michael Shurin, Clemens Esche, Jean-Marie Peron, and Michael T. Lotze, Antitumor Activities of IL-2 and Mechanisms of Action, in Chemical Immunology, IL-12, edited by L. Adorini, K. Arai, C. Berek, J. D. Capra, A.-M. Schmitt-Verhulst and B. H. Waksman, Vol. 68 at pp. 153-74, Karger, Basel, Switzerland 1997.
It is known that Granulocyte-Macrophage Colony-Stimulating-Factor (hereinafter referred to as xe2x80x9cGM-CSFxe2x80x9d) plays an essential role in induction of tumor immunity. GM-CSF is a cytokine that is made by a number of cells, including lymphocytes and it is necessary for differentiation of lineage-specific stem cells. B16 mouse melanoma cells which had been transduced with the genes for both IL-2 and GM-CSF induced stronger immunity to this tumor than any other cytokine-gene transfected Btumor cell. Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada, H., Pardoll, D., Mulligan, R. C., Vaccination With Irradiated Tumor Cells Engineered to Secrete Murine Granulocyte-Macrophage Colony-Stimulating-Factor Stimulates Potent, Specific, and Long-Lasting Anti-Tumor Immunity; Proc. Natl. Acad. Sci. USA 90: 3539-3543 (1993). The underlying molecular and cellular events are not entirely understood. It could be that GM-CSF released from the tumor cells is actively recruiting cells that are essential for primary immune responses, such as dendritic cells, and attracting these to the injection site or to the tumor cell itself. The dendritic cells then may take up antigen for presentation to T-lymphocytes attracted by cytokines released by the dendritic or other cells and/or by IL-2 released by the transfected tumor cells. Alternatively, or in addition, the dendritic cells may carry the tumor antigens to the regional lymph nodes and thereby expose the antigens to other immune system cells. It would appear that the natural immune response is more closely mimicked with vaccines incorporating the local release of two, or more, cytokines than by vaccines incorporating the release of only one cytokine.
Other experiments with IL-2 transfected tumor vaccines include those reported in Jerry A. Bash, Active Specific Immunotherapy of Murine Colon Adenocarcinoma with Recombinant Vaccinia/Interleukin-2-Infected Tumor Cell Vaccines, in Immunotherapy of Cancer With Vaccines, at pp. 331-33; E. Lord, A. McAdam, A. Felcher, M. Woods, B. Pulaski, E. Hutter, and J. Frelinger, Transfection of TGF-xcex2 Producing Tumors with IL-2 Elicits Tumor Rejection, in Immunotherapy of Cancer With Vaccines, at pp. 346-48; A. McAdam, B. Pulaski, S. Harkins, E. Hutter, J. Frelinger, and E. Lord, Coexpression of IL-2 and xcex3-IFN Enhances Tumor Immunity, in Immunotherapy of Cancer With Vaccines, at pp. 349-51. For general background on cytokine gene-transfected tumor cells see, for example, Z. Qin and T. Blankenstein, Influence of Local Cytokines on Tumor Metastasis: Using Cytokine Gene-Transfected Tumor Cells As Experimental Models, in Attempts to Understand Metastasis Formation III, Therapeutic Approaches for Metastasis Treatment, edited by U. Gxc3xcnthert, P. M. Schlag, and W. Birchmeier, pp. 55-64, Springer Verlag, Berlin 1996.
Despite the promising results with cytokine-gene-transfected cells in experimental animals, adaptation of these methods to patients faces several hurdles. As an initial matter, the technical difficulty and cost of generating sufficient quantities of gene-transfected tumor cells from a primary tumor specimen is significant. The tumor cells must be recovered from the tumor of which only a small specimen is usually available. The recovered tumor cells must be adapted to in vitro growth. This is a tedious and of ten unsuccessful procedure. The cells must be transfected in a procedure which has variable success with different tumor types and with cells of the same tumor type from different patients. See, V. W. Simons et al., Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by in vivo GM-CSF gene transfer, Cancer Research, Vol. 57 (1997) 1537-1546; R. Soiffer et al., Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony-stimulatingfactor generates potent antitumor immunity in patients with metastatic melanoma, Proceedings of the National Academy of Sciences, Vol. 95 (1998) pp. 13141-13146. (The two immediately proceeding articles will hereinafter be collectively referred to as xe2x80x9cIrradiated cancer cells and GM-CSF secretion. xe2x80x9c) Unlike tumor cells obtained from animal tumor cell lines, which are homogeneous, tumor cells recovered from a human primary cancer lesion represent an extremely heterogeneous population of cells differing in their genotypes and phenotypes. Efficiency of transfection in such a cell population can be expected to be much less than in the quasi-monoclonal animal tumor cell populations. The transfected tumor cells with the highest cytokine production rate must be identified and selected. Thus, the overall procedure is time- and cost-intensive and the results can be unpredictable.
Another problem arises because the administration of viable gene-transfected tumor cells to patients is risky and ethically unacceptable. Administration of cytokine-gene-transfected somatic cells such as fibroblasts is also risky. In animal experiments, injected cytokine-gene transfected tumor cells have occasionally lost the cytokine gene and demonstrated aggressive tumor growth, and cytokine-gene transfected fibroblasts have been shown to accelerate tumor growth. Tsai, J., Gansbacher, B., Tait, L., Miller, S. R., Heppner, G. H., Induction of Antitumor Immunity by Interleukin-2 Cene-Transduced Mouse Mammary Tumor Cells Versus Transduced Mammary Stromal Fibroblasts; J. Natl. Cancer Inst. 85: 546-52 (1993). Transfection with a cytokine or a growth factor gene has also been shown to confer a malignant or metastasizing phenotype on some tumor cells. Malik, S. T. A., Naylor, M. S., East, N., Oliff, A., Balkwill, F. R., Cells Secreting Tumor Necrosis Factor Show Enhanced Metastasis In Nude Mice; Eur. J. Cancer 26: 1031-1034 (1990).
In patient trials, tumor cells must be subjected to lethal radiation before administration to prevent replication of the tumor cells. Although the irradiated cells retain the capability of secreting the encoded cytokine, production capacity is of ten diminished. Accumulated data from various gene transfection experiments indicates that the cytokine production rate for cytokine-gene transfected cells is in the range of picograms to nanograms per 24 h per 106 cells. Colombo, M. P., Fomy, G., Cytokine Gene Transfer in Tumor Inhibition and Tumor Therapy. Where Are We Now?; Immunol. Today 15: 48-51(1994). With respect to irradiated transfected tumor cells, the combination of diminished capacity to produce the encoded cytokine and the elimination of viable, replicating cytokine-producing cells will severely limit the quantity of cytokines secreted. The contribution of replicating tumor cells to cytokine production was demonstrated by data from a study where mice were injected with IL-2-gene-transfected P815 mouse mastocytoma cells or with untransfected P815 tumor cells. Ley, V. Roth, C., Langlade-Demoyen, P. Larsson-Sciard, E. L., Kourilsky, P., A Novel Approach to the Induction of specific Cytolytic T Cells In Vivo; Res. Immunol. 141: 855-863 (1990). Within 2-3 weeks following injection of 106 untransfected cells, a tumor having a volume of about 10,000 mm3, corresponding to approximately 109-1010 tumor cells, was generated. In contrast, injection of 106 transfected cells resulted in a much slower growing tumor of only 100 mm3 volume within two weeks. This small tumor regressed within a few more weeks indicating that cytokine had been secreted not only by the initially injected 106 transfected cells but by the daughter cells as well.
Assuming a cytokine production rate of a few nanograms per 106 cells per 24 hours, it will be appreciated that the dividing tumor cells will secrete steadily increasing quantities of cytokine during the growth phase of the tumor such that, after 14 days, a cytokine production rate of a few hundred to a few thousand nanograms per 24 hours would be achieved. It will further be appreciated that, if replication of the tumor cells is prevented by irradiation the release rate of transfected X-irradiated tumor cells cannot go up during the follow up. This is in contrast to vital gene-transfected cells where there is a great dynamic in cytokine release due to the fact that the cells first multiply and produce more and more cytokine and then die and the cytokine production diminishes. Even assuming that the cytokine production rate from the initially injected cells remains stable over 14 days, the quantity of cytokines will be lower by a factor of 100 to 1,000 compared to the quantity produced by viable replicating tumor cells. In patient trials, 107 cells are typically employed in vaccines. Nevertheless, considering the large body mass of a human, it appears that the cytokine production rates obtainable from 107 irradiated non-replicating transfected tumor cells will be very low. Furthermore, it has been shown that each patient""s tumor cells show a different transfection rate and a different cytokine release rate. See, Irradiated cancer cells and GM-CSF secretion.
In sum, the use of cytokine gene-transfected cells permits the localized delivery of cytokines but it has several drawbacks which include: unpredictability of the number of gene copies introduced in the transfected cell and extent to which the gene copies are expressed; unpredictability of the amount of cytokine that will be secreted by the transfected cells; and finally possibility that the transfected tumor cells will lose the inserted gene, escape elimination and develop new tumors. In general, and despite the progress in gene therapy, the obtention of appropriately transfected cells is complicated, difficult, and expensive.
Notwithstanding the intense effort expended in research directed to the preparation of tumor vaccines capable of stimulating immune response to specific tumor antigens, none have been developed which are simple, reliable and relatively inexpensive. As a result, efforts to develop such vaccines continue unabated.
The fight against infectious diseases with vaccines also teaches that prevention of infectious diseases with vaccines is easier than therapy of the same diseases under development. This experience has been interpreted as suggesting that prophylactic vaccination against cancer may be more successful than vaccination when the disease is at an advanced stage. Immunotherapy of Cancers With Vaccines, at p. 4. However, therapeutic vaccination may be the only resort to fight against certain diseases and in particular against tumors.
In light of the problems or drawbacks associated with the systemic administration of cytokines or with the use of cytokine gene-transfected cells, it is desirable to provide a composition and method of administration for immunological treatment of tumors with a selected cytokine whose non-systemic administration leads to positive effects on the treatment of tumors. Such preparation has to be administered at a physiologically acceptable dosage by an appropriate administration route. It is also desirable to provide a composition and method of administration for immunological treatment of tumors with properties that include: (a) good tolerance with the receiving living being, a property that requires the absence of or at least minimization of undesirable side effects; (b) high effectivity upon administration in amounts of the order of xcexcg rather than in the order of mg as in systemic treatments; (c) appropriate form for local administration; and (d) appropriate form for administration in a controlled and predictable manner.
An object of the present invention is the induction of immune responses to tumors. In particular, an object of the present invention is the design of a depot formulation including an immunostimulant such as a cytokine that is optimally suited as an adjuvant for the induction of cell-dependent cytotoxic immune responses to cellular antigens.
Another object of the present invention is to provide methods of preparing tumor vaccines capable of stimulating immune responses to tumor cells which are simple, reliable, and relatively inexpensive to use.
Another object of the present invention is to provide a composition comprising antigenic material and a depot with immunostimulating material that is tolerated by living organisms without unacceptably deleterious side effects. In particular, an object of the present invention is to provide a composition with antigenic tumor cells, preferably inactivated cells, and a depot with at least one cytokine that is tolerated by living organisms without unacceptably deleterious side effects.
Another object of the present invention is to provide tumor vaccines comprising tumor cells mixed with immunostimulant adsorbed to aluminum hydroxide in the same inoculum such that active specific immunity is induced.
Another object of the present invention is to provide an intratumoral treatment preparation including an immunostimulant adsorbed to a depot which can be injected into a tumor.
A feature of the compositions and methods of this invention is that the compositions can be administered as vaccines are typically administered, and also they can be administered in intratumoral applications. It is also a feature of the compositions of the present invention that the specific characterization of treatment is not critical to the beneficial results derived therefrom.
These and other objects and features of the present invention will become more fully apparent from the following description and appended claims, or may be learned by the practice of the invention as set forth hereinafter.
To achieve the foregoing objects, and in accordance with the invention as exemplified by embodiments described herein, compositions and methods are provided which can be utilized in active immunization as a prophylactic treatment or a therapeutic treatment for tumors. The compositions are employed as injectable tumor vaccines or as preparations for intratumoral administration and are capable of stimulating immune responses to specific tumor antigens.
The tumor vaccines induce an immune response to prevent tumor proliferation and growth and include an antigenic cellular material comprising a plurality of inactivated tumor cells or tumor cell portions, an adsorbent depot material, and at least one immunostimulant adsorbed to the depot material. The tumor cells or tumor cell portions are inactivated such that the antigenic structures thereof are preserved, with the tumor cells or tumor cell portions being capable of inducing an immune response. The depot material with absorbed immunostimulant is mixed with the tumor cells or tumor cell portions to form the vaccine compositions. The preparations for intratumoral administration include the depot material adsorbed immunostimulant without the antigenic cellular material. The immunostimulant adsorbed to the depot material permits release of biologically active quantities of the immunostimulant over a period of time rather than all at once.
A preferred immunostimulant for the compositions of the invention is a cytokine, such as a recombinant human cytokine, particularly a lymphokine, and more particularly interleukin-2. Other useful immunostimulants besides interleukin-2 include interleukin-4, interleukin-12, G-CSF, GM-CSF, and combinations thereof, as well as bacterial cell wall components such as muramyl dipeptide. Preferably, the immunostimulant is present in the compositions in an amount of at least about 10 xcexcg.
A preferred depot material is an aluminum-based depot such as aluminum hydroxide, although other inorganic materials such as calcium phosphate as well as other inorganic or organic particles which are capable of adsorbing proteins such as latex particles and ion exchangers can also be used as depot material, along with various combinations thereof. In the context of this invention, the cytokine adsorbed to the depot material is, in combination, an adjuvant.
The ratio of immunostimulant to depot material in the compositions of the invention is chosen such that the depot material adsorbs all of the immunostimulant. Preferably, the ratio of immunostimulant to depot material is from about 0.1 to about 1 xcexcg/xcexcg.
A preferred treatment preparation for intratumoral injection to eliminate a tumor and induce an immune response to the tumor includes an inorganic depot material composed of aluminum hydroxide, and a cytokine immunostimulant adsorbed to the depot material.
In a method for inducing an immune response to prevent tumor proliferation and growth, one or more tumor vaccine inoculums according to the invention are provided, and an effective amount of one or more of the tumor vaccine inoculums are injected at least once so as to permit release of biologically active quantities of the immunostimulant over a period of time to induce an immune response to the presence of active tumor cells. The tumor cells which can be used in the vaccine inoculums may be obtained from various sources such as tumor cells recovered from a primary lesion or secondary lesions in a patient, tumor cells prepared by in vitro culture of the tumor cells recovered from a patient, tumor cells prepared by in vitro culture of allogeneic tumor cell lines, and combinations thereof. The tumor cell portions which can be used in the vaccine inoculums may be obtained from various processes such as lyzing of the tumor cells, preparing extracts of cell membranes and membrane vesicles, and combinations thereof.
The tumor vaccine inoculum can be injected to prevent proliferation and growth of various tumors such as melanoma, renal carcinoma, prostate carcinoma, colon carcinoma, pancreas carcinoma, and lung carcinoma, as well as B lymphoma. It should be understood that a particular vaccine composition will contain tumor cells that correspond to the particular tumor being treated. Thus, a vaccine composition for renal carcinoma will contain renal carcinoma cells or cellular portions thereof. The tumor vaccine inoculums can be injected into a patient subcutaneously and/or intradermally.
In a preferred method for intratumoral treatment, a treatment preparation is provided as described above, and an effective amount of the treatment preparation is injected at least once into a tumor so as to permit release of biologically active quantities of the cytokine immunostimulant over a period of time to induce an immune response.
Instead of relying on systemic administration of cytokines or on cytokine gene-transfected cells, the present invention encompasses the following strategy. In-situ cytokine-producing gene-transfected cells are characterized by having the source of cytokine and the source of immunogenic material combined in one entity. Instead of having this combination in a gene-transfected cell, the present invention uses mixtures of tumor cells and cytokines in depot formnulations. One of the advantages of this procedure is that it is relatively easy to prepare vaccines from irradiated tumor cells and one or several cytokines in depot formulations. The cytokine depots can be kept on the shelf and used as needed. It is possible and easy to vary the dose of both components, the tumor cells and the cytokine, thus being able to elucidate their contribution to the induction of the immune response and to make vaccines with optimal properties for each patient. Another advantage of this invention is that parameters that characterize the vaccine composition and its administration can be elaborated on a quantitative basis rather than by trial and error. These parameters include the composition of the vaccine, and more specifically the tumor cell dose and cytokine dose, and the vaccination conditions, including vaccination site and vaccination course.